GoKawiilRNA sequencing and analysis
E. coli BL21(DE3) cells (NEB: C2527H), which lack endogenous copies of IS621 elements, were transformed with plasmids encoding either the RE–LE junction or the LE–recombinase–RE region of the IS621 element. The cells were plated on LB (lysogeny broth) agar with kanamycin and grown overnight at 37 °C. Colonies were scraped from the plates, and RNA was extracted using a Direct-zol RNA miniprep kit (Zymo Research). To assess the expression of the IS621 elements encoded in the E. coli genome, RNA was extracted using a Direct-zol RNA miniprep kit from One Shot E. coli Mach1 T1R cells (F– φ80lacZΔM15 ΔlacX74 hsdR(r K– , m K+ ) ΔrecA1398 endA1 tonA) (Thermo Fisher Scientific, C862003) harvested from bacterial plates 24 h after plating at 37 °C.
RNA-seq was carried out on a NovaSeq X Plus sequencer in collaboration with Novogene Research Services (Novogene). Paired-end RNA-seq reads from each sample were aligned to the respective plasmid and genomic reference sequences using Burrows–Wheeler Aligner-MEM17. Aligned reads were filtered using SAMTools18 for reads that originated from the strand that encodes the bRNAs. For each locus of interest on the reference sequences, we used the Pysam Python module to count the coverage depth at the single-base level from these filtered reads, with a quality threshold of 0. As the E. coli Mach1 genome has three separate IS621 loci that are highly homologous with one another, many of the RNA-seq reads that mapped to these regions were expected to lack unique mapping to one of the three loci. Thus, among the reads that were mapped to any of the IS621 loci, pairs of reads in which either pair had a mapping quality of zero (meaning the read can map equally well to other parts of the reference sequence) were classified as ‘nonspecific’ reads, and the coverage from these reads was counted separately from the reads that mapped specifically to one of the three IS621 loci.
Detection of plasmid-based IS621 excision by PCR
Plasmids containing elements with the wild-type bRNA (pre-TSE for excision) or the engineered bRNA (post-TSE for excision) were transformed into E. coli BL21(DE3), which lacks endogenous copies of IS621 elements, ensuring that all detected excision events originate from the plasmid-borne construct. For constructs lacking a T7 promoter upstream of the bRNA, bacteria were plated on LB agar with kanamycin and grown overnight at 37 °C. For constructs with a T7 promoter, plates were supplemented with 0.07 mM IPTG. Colonies were scraped from plates, and plasmid DNA was extracted using a QIAprep Spin Miniprep Plus Kit (Qiagen). Primers were designed to span the post-excision (LT–RT) junction site left after element excision from the plasmid and yield a 110-bp product. The same primers used to detect circularized IS621 intermediates from the Mach1 genome were also used to confirm the production of circular intermediates from plasmid-borne constructs, yielding a 725-bp product. All PCRs were performed with the same protocol described below for the detection of native IS621 excision and were fractionated on 2% agarose gels and visualized with SYBR Gold (Thermo Fisher Scientific). All PCR products were sequence-verified by Sanger sequencing.
Detection of circular intermediates and post-excision sites
Genomic DNA was isolated from freshly purchased aliquots of E. coli strains in liquid culture using Zymo Quick DNA Miniprep Plus Kits (Zymo), according to the instructions of the manufacturer. Whole-genome sequencing was performed at 100× coverage using the Plasmidsaurus whole-genome nanopore sequencing service, which confirmed that the One Shot Mach1 E. coli T1R strain harbours three copies of IS621, whereas the E. coli BL21(DE3) strain contains zero copies. Bacteria were plated on LB agar and grown overnight at 37 °C. Colonies were then scraped from the plates, and total DNA was extracted using a Zymo Quick DNA Miniprep Plus Kit. Primers were designed to match all three IS621 recombinase-coding sequences in the Mach1 genome, such that PCR amplification would occur only on excised and circularized IS621 sequences from any locus. Moreover, primers were designed to base pair with the junction remaining post-excision at all three loci encoding IS621, such that amplification with a downstream primer would yield a specific product. All PCRs were performed using Platinum SuperFi 2× MasterMix (Thermo Fisher Scientific) with the following protocol: 98 °C for 120 s; 25 cycles of 98 °C for 10 s, 65 °C for 15 s and 72 °C for 15 s; and 72 °C for 2 min. PCR products were subsequently fractionated on 2% agarose gels and visualized with SYBR Gold.
In vitro recombination measurement by qPCR
DNA substrates (51-bp LH, 121-bp RH, 86-bp tDNA and 86-bp dDNA) were purchased from IDT as ssDNA and annealed by heating to 95 °C followed by slow cooling to 4 °C over 1 h in a thermocycler. DNA substrates (0.25–0.5 µM) were mixed with the IS621–bRNA complex (10 µM) in 20 µl buffer containing 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM MgCl 2 and 1 mM dithiothreitol (DTT), and then the reactions were incubated at 37 °C for 2 h. The reactions were quenched by the addition of 50 mM EDTA, treated with 8 µg RNase A (NEB) at 50 °C for 1 h, and then treated with 3 units of Proteinase K at 37 °C for 1 h. After RNA and protein digestion, the DNA was purified using DNA Clean & Concentrator-5 (Zymo Research) and eluted with 75 °C nuclease-free water. For qPCR analysis, reaction dilutions were prepared, and qPCR was performed on a LightCycler 480 II instrument (Roche) using primers and PrimeTime qPCR probes (final concentration 0.5 µM) purchased from IDT, along with TaqMan Fast Advanced qPCR mix. In vitro recombination products are expected to have an unligated bottom strand, with the two nucleotides adjacent (3′) to the core mismatched with the top strand, after the bottom strand exchange step of the reaction mechanism. Therefore, to accurately quantify the reaction efficiency, considering the polymerase processivity through lesions and the primer binding near nicks, qPCR signals for different reactions were compared with standard curves generated using known quantities of 121-bp RH (LD–RT, insertion product) DNA and 86-bp target (LT–RT, excision product) DNA containing an unligated bottom strand with the expected mismatches adjacent to the core. These standards were prepared by annealing the top strand of RH/target (121/86 nt) with two oligos (56- and 65-nt RH and 56- and 30-nt target) constituting the bottom strand.
In vivo excision efficiency measurement by qPCR
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